The present invention relates to novel enteropeptidase (EP) variant polypeptides derived from Japanese Medaka (Oryzias latipes). More particularly, the present invention relates to novel EP variant polypeptides with enhanced substrate specificity, polynucleotides encoding the EP polypeptides, nucleotide constructs, vectors and host cells comprising the polynucleotides, methods for producing the polypeptides and polynucleotides, and kits.
EP (enterokinase, EC 3.4.21.9) is a heterodimeric glycoprotein present in the duodenal and jejunal mucosa and is involved in the digestion of dietary proteins. Specifically, EP catalyzes the conversion, in the duodenal lumen, of trypsinogen into active trypsin via the cleavage of the acidic propeptide from trysinogen (Light et al., Trends Biochem. Sci., 14:110-112 (1989)). The activation of trypsin initiates a cascade of proteolytic reactions leading to the activation of many pancreatic zymogens, including chymotrypsinogen, proelastase, procarboxypeptidases, and some prolipases (Grishan et al., Gastroenterology, 85:727-731 (1983)).
To date, studies have reported the molecular cloning of EP from several mammalian sources, including cattle (LaVallie et al., J. Biol. Chem., 268:23311-13317 (1993); Kitamoto et al., Proc. Natl. Acad. Sci. USA, 91:7588-7592 (1994)), humans (Kitamoto et al., Biochemistry, 34:4562-4568 (1995)), pigs (Matsushima et al., J. Boil. Chem., 269:19976-19982 (1994)), rats (Yahagi et al., Biochem. Biophys. Res. Commun., 219:806-812 (1996)), and mice (Yuan et al., Am. J. Physiol., 274:342-349 (1998)). These studies provided much information on the structural details and organization of EP, and opened a path to further investigation of the molecular properties of this protease. For example, it was reported that the N-terminal heavy-chain is required for efficient activation of trypsinogen by the serine protease domain of the C-terminal light chain (Lu et al., J. Biol. Chem., 272:31293-31300 (1997); Mikhailova et al., FEBS Lett., 442:226-230 (1999)). In addition, a recent study by Lu et al. established the tertiary structure of the bovine EP catalytic domain, thereby demonstrating that Lys99, which is situated in a unique exosite on the enzyme surface, involves in the specific cleavage of trypsinogen and similar peptidyl substrates (Lu et al., J. Mol. Biol., 292:361-373 (1999)). A more recent study reported that a mucin-like domain found in the heavy chain of EP can be a targeting signal for apical sorting of the protein (Zheng et al., J. Biol. Chem., 277:6858-6863 (2002)).
EP is highly specific for the sequence Asp-Asp-Asp-Asp-Lys (D4K) (SEQ ID NO: 1) of trypsinogen (Bricteux-Gregoire et al., Comp. Biochem. Physiol., 42B: 23-39 (1972)). It is generally believed that EP (or enteropeptidase-like enzyme) is present in all vertebrates. This belief comes from the finding that in almost all vertebrate species a short peptide sequence of Asp-Asp-Asp-Asp-Lys (D4K) (SEQ ID NO: 1) is found in the presumed activation site of trypsinogens (14). However, no information on EP in vertebrates other than mammals has been made available to date. EP is highly specific for the sequence Asp-Asp-Asp-Asp-Lys (D4K) (SEQ ID NO: 1) of trypsinogen (Bricteux-Gregoire et al., Comp. Biochem. Physiol., 42B:23-39 (1972)). Because of the high degree of D4K (SEQ ID NO: 1) specificity, EP has been used as a suitable reagent for cleaving substrate proteins. Indeed, bovine EP has been widely used for this purpose (Collins-Racie et al., Biotechnology, 13:982-987 (1995)).
Nonetheless, the conventional system utilizing bovine EP still has significant drawbacks for industrial application due to its nonspecific proteolytic activity. More particularly, while bovine EP protease cleaves at the EP-cleavage site of recombinant fusion proteins, it also simultaneously hydrolyzes other peptide bonds of the proteins to a considerable degree because of its nonspecific proteolytic activity. This causes a seriously low yield of the targeted protein. Such nonspecific activities of bovine EP also can be an obstacle in the preparation of active recombinant proteases where the EP is employed for cleavage of the inactive fusion protein. This is particularly serious when the proteases to be examined are ones with very low activity for synthetic and naturally occurring protein substrates. In addition, such nonspecific activities of bovine EP make it difficult to determine whether the target recombinant proteases have been successfully activated.
Hence there is a need to generate a novel EP variant polypeptide that substantially lacks nonspecific proteolytic activity while retaining its high specificity for D4K sequence (SEQ ID NO: 1).